Silent cerebral infarctions (SCI) occur in 22% of children with sickle cell disease (SCO), are associated with decreased cognitive function and are a significant risk factor for the development of stroke. By definition, these children have MRI and pathologic evidence consistent with cerebral infarction, without overt symptoms of stroke. As the etiology of SCI is unknown, we lack the means to easily identify children with SCI or at risk for SCI to guide early intervention or follow the success of therapy. The overall goal of this grant proposal is to identify diagnostic/etiologic plasma biomarkers of SCI in children with SCO. In our pilot studies, using a non-biased proteomic analysis of plasma samples from patients with or without SCI in the SIT Trial, we have found significant differences in the plasma protein profile of children with SCI and have identified two circulating brain specific proteins, neural cell adhesion molecule 1 and glial fibrillary acidic protein, a known biomarker of stroke. Based upon these studies, we propose to identify the plasma biomarkers of SCI, establish whether circulating biomarker proteins can predict SCI and probe deep in the plasma proteome of children with SCI to identify diagnostic biomarkers validated to the brain and vasculature. The significance of the proposed studies is that identification of circulating markers of SCI would allow early intervention in these children and provide insight into the design of new therapies. This is particularly important, as our present therapy of repeat transfusions carries a high morbidity in and of itself. Therefore, we hypothesize that plasma samples from children with SCO and SCI will contain diagnostic/etiologic biomarkers of SCI. To examine this hypothesis, our proposal draws on the unique resources of the large and highly phenotyped plasma samples in the SIT Trial Biologic Respository and the NHLBI Cardiovascular Proteomics Center at Johns Hopkins. In three aims we will: first, using state of the art non-biased proteomic techniques (HPLC. ITRAQ and LC/MS/MS), identify plasma diagnostic biomarkers of SCI;second, validate our biomarkers using a combination of western blotting and high throughput multiplex antibody arrays against the SIT Trial plasma repository to test the efficacy of the biomarkers in predicting SCI;and third, go back to the brain, to localize biomarker proteins to the brain and test their specificity for SCO and ischemic neurologic injury. These studies describe the discovery and validation phases of work to uncover diagnostic/etiologic plasma biomarkers of SCI in children with SCO. It is also possible that these studies may provide new insights into the development of stroke in SCO and stroke in general.